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PubMed2001

Expression of matrix metalloproteinases during ascorbate-induced differentiation of osteoblastic MC3T3-E1 cells.

The mouse calvarial osteoblast MC3T3-E1 cells released 92 kDa and 68 kDa of gelatinase activities into the conditioned media (CMs) from undifferentiated cells. When differentiation was induced by cultivating cells with ascorbate-2-phosphate (AscP), 68-kDa activity increased significantly in parallel with production of 60-kDa activity. These enzymes required Ca2+ and Zn2+ ions for their proteolytic activities. The 68-kDa activity was immunologically identified as latent matrix metalloproteinase 2 (MMP-2). The 92-kDa activity was deduced to be latent MMP-9 based on its molecular mass. The 60-kDa activity band was found to possess both gelatin and beta-casein hydrolyzing activities, indicating that this activity band might comprise the active form of MMP-2 and latent MMP-13. MC3T3-E1 cells were found to express MMP-2, MMP-13, and membrane type (MT)1-MMP genes by Northern blotting. MMP-2 was expressed constitutively. MMP-13 was up-regulated during the growth with AscP. MT1-MMP expression also was modulated by AscP; at the early stage of differentiation, its messenger RNA (mRNA) level increased and then decreased gradually to the control level. These changes in the profiles of MMPs observed here could be attributed to the maturation of collagenous extracellular matrix (ECM) induced by AscP.

3T3 Cells↗
PubMed1999

A unique talin homologue with a villin headpiece-like domain is required for multicellular morphogenesis in Dictyostelium.

Molecules involved in the interaction between the extracellular matrix, cell membrane and cytoskeleton are of central importance in morphogenesis. Talin is a large cytoskeletal protein with a modular structure consisting of an amino-terminal membrane-interacting domain, with sequence similarities to members of the band 4.1 family, and a carboxy-terminal region containing F-actin-binding and vinculin-binding domains [1] [2]. It also interacts with the cytoplasmic tail of beta integrins which, on the external face of the membrane, bind to extracellular matrix proteins [3]. The possible roles of talin in multicellular morphogenesis in development remain largely unexplored. In Dictyostelium, a eukaryotic microorganism capable of multicellular morphogenesis, a talin homologue (TALA) has previously been identified and shown to play an important role in cell-to-substrate adhesion and maintenance of normal elastic properties of the cell [4] [5] [6]. Here, we describe a second talin homologue (TALB) that is required for multicellular morphogenesis in the development of Dictyostelium. Unlike any other talin characterised to date, it contains an additional carboxy-terminal domain homologous to the villin headpiece.

Amino Acid Sequence↗
PubMed1996

Photolysis of Matrix-Isolated 4-R-1,2,4-triazoline-3,5-diones: Identification of Aziridine-2,3-dione Transients.

Matrix-isolated 4-methyl-1,2,4-triazolinedione 1a and 4-phenyl-1,2,4-triazolinedione 1b were photolyzed at lambda >/= 335 nm and lambda >/= 310 nm, respectively. The reactions induced by photolysis were monitored by FT-IR spectroscopy. The isocyanates 2a and 2b are always the more abundant products with carbon monoxide. Methyl- and phenylaziridine-2,3-diones (3a and 3b) were detected as minor, but well-identified reaction products. The IR experimental absorption bands were assigned by comparison with literature data and with simulated infrared spectra obtained by ab initio calculation at the 6-31G level. Stable at the matrix temperature (10 K), 3a and 3b photolyzed to isocyanates and CO when irradiated at lambda >/= 230 nm. Irradiation of 2b at this wavelength induces its decomposition. The kinetic data show that the rate constant process 1a --> 2a is faster than the 1a --> 3a process (3.29 x 10(-)(3 )and 2.35 x 10(-)(4)min(-)(1) respectively).

Journal Article↗
PubMed1990

A unified model of eukaryotic chromosomes.

A revised model of DNA packaging into chromosomes is presented. Its features are consistent with observed structural dimensions and the molecular periodicities related to transcription, replication and matrix attachment domains. The transitions between euchromatic, heterochromatic and metaphase states are explained simply. Molecular and physical properties of chromosomal bands, and their correlation with specific DNA sequence motifs are discussed.

Animals↗
PubMed1994

Hyaluronan receptor (CD44) expression and function in human peripheral blood monocytes and alveolar macrophages.

CD44 glycoproteins are present on the surfaces of many hematopoietic cells and in some cases can bind hyaluronan, a major component of the extracellular matrix. In the present study, we have found that newly explanted human peripheral blood monocytes (PBMs) exhibit a major CD44 band of 85 kDa, whereas autologous alveolar macrophages (AM phi) express multiple isoforms ranging from 85 to 200 kDa. Within 4 h in culture, PBMs began expressing new CD44 isoforms of 120, 150, and 180 kDa. Newly explanted AM phi specifically bound [3H]hyaluronan (135 cpm/microgram protein), but newly explanted PBMs did not. However, in vitro cultured PBM progressively acquired the ability to bind [3H]hyaluronan and exhibited specific binding of hyaluronan similar to that of AM phi (113 cpm/microgram protein) after 4 days in culture. In both case, the binding of [3H]hyaluronan was specifically inhibited by the addition of monoclonal antibody directed against CD44. AM phi readily degraded [3H]hyaluronan and reached a plateau after 4 days in culture (115 cpm/microgram protein). Newly explanted PBM exhibit no hyaluronan degradation and only a small degradative activity after 4 days in culture (6 to 11 cpm/microgram protein). Thus, CD44 expression and function appear to change as PBM mature in vitro resembling more that found in AM phi.

Adult↗
PubMed1994

Demonstration of glutathione peroxidase in rat liver peroxisomes and its intraorganellar distribution.

Earlier, we reported that rat liver peroxisomes contain Cu-Zn superoxide dismutase (J. Biol. Chem. 267, 6870), thereby suggesting a new antioxidant role for this organelle in free radical metabolism. In this study, we report for the first time that mammalian peroxisomes also contain glutathione peroxidase. Using highly purified rat liver peroxisomes isolated by Nycodenz gradient, we found that peroxisomes contain glutathione peroxidase which shows enzymatic activity with different substrates such as hydrogen peroxide, cumene hydroperoxide, and t-butyl hydroperoxide. This activity could be inhibited in vitro by mercaptosuccinate. Western blot analysis revealed that peroxisomes from control and ciprofibrate-treated livers show immunoreactive bands with antibodies raised against glutathione peroxidase. The intraperoxisomal distribution of glutathione peroxidase was investigated by using peroxisomal membrane and matrix proteins. The results revealed that glutathione peroxidase is a matrix enzyme. The presence of glutathione peroxidase in peroxisomes provides an alternate enzyme system responsible for the degradation of organic peroxides and the degradation of H2O2 under conditions in which catalase is inactivated (e.g., ischemia-reperfusion and endotoxemia). These findings suggest that glutathione peroxidase in peroxisomes may play a novel role in the cellular antioxidant responses to various oxidative stress conditions.

Animals↗
PubMed1998

Line Intensities for the 8-µm Bands of SO2

Using both high-resolution (R = 0.003 cm-1) and medium-resolution (R = 0.12 cm-1) Fourier transform spectroscopy, it has been possible to measure accurately a large set of individual line intensities for the nu1 and nu3 bands of SO2 in the 950-1400 cm-1 spectral region. These intensities were introduced into a least-squares fit calculation allowing one to obtain the expansion of the transition moment operator of the nu1 and nu3 bands. For these intensity calculations, the theoretical model takes into account the vibration-rotation interactions linking the upper levels involved in the nu1, 2nu2, and nu3 interacting SO2 bands. Finally, a synthetic spectrum of the 8-µm SO2 bands has been generated using the dipole moment expansion determined in this work and the molecular parameters and the Hamiltonian matrix given in a previous analysis. Copyright 1998 Academic Press.

Journal Article↗
PubMed1991

New method of ultrastructural localization of WGA-binding sites in the rabbit cornea using quick-frozen specimens followed by freeze-substitution.

Wheat germ agglutinin (WGA) conjugated with apo-horseradish peroxidase-gold was used as a probe for ultrastructural localization of N-acetyl-glucosamine and N-acetyl-neuraminic acid residues of glycoconjugates in posterior stroma and Descemet's membrane of rabbit corneas. Ultra-thin sections of quick-frozen, freeze-substituted, and Epon or Lowicryl K4M-embedded tissue were used. Numerous WGA-binding sites were detected in the stromal matrix and Descemet's membrane in both embedding procedures. Labelling was more intense with low-temperature embedding in Lowicryl K4M than with Epon. Gold particles preferentially labelled the major collagen fibrils of the stroma but otherwise appeared uniformly distributed over both extracellular matrices, except in some areas of the stromal matrix that were barely labelled. Frequently, the inter-particle distance of gold particles on collagen fibrils corresponded to the banding period of type I collagen fibrils, suggesting that they point out keratan sulphate proteoglycans which contain WGA-binding sites. The combination of cryotechniques and post-embedding procedures may be very effective for lectin histochemistry of the cornea and other eye tissues.

Acetylglucosamine↗
PubMed2004

Modelling and simulation of acoustic wave propagation in locally resonant sonic materials.

Sonic crystals are artificial structures consisting of a periodic array of acoustic scatterers embedded in a homogeneous matrix material, with a usually large impedance mismatch between the two materials. They exhibit strong sound attenuation at selective frequency bands due to the interference of multiply reflected waves. However, sound attenuation bands in the audible range are only achieved by unfunctionally large sonic crystals. If local resonators are used instead of simple scatterers, the frequencies of the attenuation bands can be reduced by about two orders of magnitude. In the present paper we perform numerical simulations of acoustic wave propagation through sonic crystals consisting of local resonators using the local interaction simulation approach (LISA). Three strong attenuation bands are found at frequencies between 0.3 and 6.0 kHz, which do not depend on the periodicity of the crystal. The results are in good qualitative agreement with experimental data. We analyze the dependence of the resonance frequencies on the structural parameters of the local resonators in order to create a tool for design and optimization of any kind of sonic crystal.

Journal Article↗
PubMed2003

Identification of a key protein associated with cerebral ischemia.

The biochemical effects of permanent focal ischemia following unilateral occlusion of the middle cerebral artery in rats were studied by determining the content of specific proteins of the affected areas in the cerebral hemisphere. Brain proteins were prepared 72 h after the occlusion and analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis. A significant increase in 66 and 80 kDa components and a paradoxical decrease in 260 kDa protein occurred in the ischemic brain tissues. The 66 and 80 kDa protein bands were identified as albumin and transferrin, respectively. The 260 kDa protein was analyzed by peptide mass fingerprinting (PMF) and matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS). The isoelectric point of the 260 kDa protein was 4.65 determined by isoelectric focusing. The data obtained from PMF were used in searching the protein database for homologous components. Three proteins with partial homology were identified. They were the microtubule-associated protein 1A, protein-tyrosine phosphatase zeta precursor (phosphacan), and protein kinase A anchoring protein 6. Polyclonal antibodies against the 260 kDa protein were raised and used to immunolocalize the antigen in various tissues. Positive staining occurred with brain neurons and pyramidal cells, islet cells, podocytes of kidney glomeruli, and endothelial cells of the venous sinuses of the spleen. The localization of 260 kDa protein strongly implies its function in these tissues. Its physiological and pathophysiological significances need to be clarified in future.

Animals↗
PubMed2004

[Proteomic analysis associating two-dimensional electrophoresis and mass spectrometry to identify lacrimal proteins: a case study].

PURPOSE: Identification of a lacrimal protein by proteomic analysis, i.e., two-dimensional electrophoresis and mass spectrometry. MATERIAL AND METHODS: We studied the tears of a 25-year-old female with adrenal gland hyperplasia and hyperandrogenism complaining of chronic dryness and mild bilateral papillary hypertrophy. An allergologic workup was negative. Agarose electrophoresis of the tears showed a bilateral high level of rapid migrated proteins. RESULTS: Dodecyl sulfate polyacrylamide gel electrophoresis of the tears from both eyes showed a highly stained 15-kDa band after Coomassie colloidal blue coloration compared to controls. On two-dimensional electrophoresis, this band focused on a single spot at pI 7.0. After tryptic digestion in gel, peptide mass fingerprint analysis by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry provided clear identification of cystatin SN. It is known that mRNA regulated by androgens and encoding glycoproteins homologous to human cystatin exists in the rat lacrimal gland. CONCLUSION: We conclude that the hyperandrogenism of the patient may be cause for the hypersecretion of this cystatin SN, giving an explanation for the high level of rapid migrated proteins (lipocalins). This result provides a concrete example of the proteomic tool used to identify lacrimal proteins, still largely unknown.

Adult↗
PubMed1999

Comparison between site-specific DNA binding proteins of male and female Schistosoma mansoni.

Several amplicons with approximately 120 bp each, obtained from the upstream domain of Schistosoma mansoni female-specific gene F-10, were coupled to Dynabeads M-280 streptavidin. The beads were used as a matrix for affinity purification of nuclear proteins obtained from mixed populations of adult worms. A protein of approximately 12 kDa, bound to the DNA in a sequence-independent manner. In contrast, when the DNA matrix was narrowed down to smaller synthetic oligonucleotides, bearing sequences corresponding to the TATA box and the CAAT box, band-shift assays revealed that different nuclear proteins from either adult male or female worms formed complexes with the DNA adduct. In order to characterise the bound proteins, the same oligonucleotides were UV cross-linked to the male and female protein extracts. Whilst the band shift experiments showed that the proteins from each sex produced a distinct mobility pattern when the TATA box sequences were tested and a similar one when the CAAT box sequences were added to the proteins, UV cross-linking experiments revealed clear qualitative differences between both, male and female proteins and also between the proteins binding to the two motifs. These results are compatible with a model in which the differential expression of the F-10 gene might depend on individual sub-sets of proteins.

Animals↗
PubMedDate not supplied

Two-dimensional/ATR infrared correlation spectroscopic study on water diffusion in a poly(epsilon-caprolactone) matrix.

In the present contribution, two-dimensional ATR-FTIR spectroscopy was used to study the diffusion of water in poly(epsilon-caprolactone) (PCL). In the spectral region of the nu(OH) stretching vibration of water, four absorption bands (3635, 3560, 3411, and 3220 cm(-1)) can be identified. The higher wavenumber band pair at 3635 and 3560 cm(-1) is assigned to the antisymmetric and symmetric OH stretching vibrations, respectively, of water which is partially hydrogen-bonded to the carbonyl groups of PCL, whereas the lower frequency band pair at 3411 (antisymmetric) and 3220 cm(-1) (symmetric) is attributed to the OH stretching vibrations of bulk water which is fully hydrogen-bonded to other water molecules. From the asynchronous map of a 2D correlation analysis of spectra recorded during the diffusion of water into PCL, it was concluded that during the diffusion process the water molecules first penetrate into the free volume (microvoids) of the PCL matrix or are molecularly dispersed in the polymer matrix and then form hydrogen bonds with the C=O groups of the polymer.

Biocompatible Materials↗
PubMed1997

Luminescent and Redox-Active Iridium(III)-Cyclometalated Compounds with Terdentate Ligands.

Two novel bis-terdentate Ir(III)-cyclometalated complexes, [Ir(L1)(L1(-))](2+) (1) and [Ir(L1(-))(2)](+) (2), have been prepared (L1 is 2,6-bis(7'-methyl-4'-phenyl-2'-quinolyl)pyridine; L1(-) is its mono-anion, see Figure 1). To the best of our knowledge, 1 and 2 are the first luminescent and redox-active Ir(III)-cyclometalated bis-terdentate compounds. In acetonitrile solution, on oxidation, 2undergoes a reversible, metal-centered, one-electron oxidation at +1.40 V, whereas 1 does not exhibit any oxidation process up to +2.00 V. On reduction, both compounds undergo four reversible ligand-centered one-electron processes. The absorption spectra of the compounds are dominated by moderately intense (epsilon in the 10(3)-10(4) M(-)(1) cm(-)(1) range) spin-allowed metal-to-ligand charge-transfer (MLCT) bands at wavelengths longer than 350 nm and by intense (epsilon in the 10(4)-10(5) M(-)(1) cm(-)(1) range) ligand-centered (LC) bands at shorter wavelengths. The complexes exhibit an intense luminescence both at 77 K in MeOH/EtOH, 4:1 (v/v), rigid matrix (1, lambda(max) = 592 nm, tau = 20 &mgr;s; 2, lambda(max) = 598 nm, tau = 9 &mgr;s) and at room temperature in deoxygenated acetonitrile solution (1, lambda(max) = 620 nm, tau = 325 ns, Phi = 0.005; 2, lambda(max) = 630 nm, tau = 2.3 &mgr;s, Phi = 0.066). In all cases, the emission is assigned to triplet MLCT levels (namely, Ir --> L1 and Ir --> L1(-) in 1 and 2, respectively).

Journal Article↗
PubMed2005

Photoisomerization of cis-1-(2-naphthyl)-2-phenylethene in methylcyclohexane at 77 K: no hula-twist.

The cis-trans photoisomerization of cis-1-(2-naphthyl)-2-phenylethene (c-NPEA and c-NPEB) was studied in methylcyclohexane glass at 77 K. The progress of the reaction was followed by fluorescence spectroscopy. Formation of the extended trans conformer (t-NPEB) is revealed by the growth of sharp vibronic bands on the broad structureless fluorescence spectrum of c-NPE. Principal component analysis with self-modeling of a matrix consisting of spectra generated by irradiation of c-NPE at different excitation wavelengths and spectra of t-NPE measured under the same conditions as a function of excitation wavelength reveals that the diabatic photoisomerization is a completely conformer-specific one-bond twist process: c-NPEB --> t-NPEB. These observations are consistent with observations in solution at ambient temperature. They demonstrate unequivocally that free volume restrictions imposed by the amorphous glassy environment do not open a hula-twist process.

Cold Temperature↗
PubMed2004

Photolysis of 3-(2-Formylphenyl)-3-chlorodiazirine in an Ar matrix at low temperature.

Photolysis of 3-(2-formylphenyl)-3-chlorodiazirine in an Ar matrix at 13 K was monitored by IR and UV-vis spectroscopies. Characterization of the products was carried out by comparing the spectra with the theoretical IR spectra obtained by using DFT, which suggested that anti,anti-2-formylphenyl(chloro)carbenes (a,a-2), ketenes (syn- and anti-3), chlorobenzocyclobutenone (4), and chloroisobenzofurane (5) were formed. This indicates that carbene 2 interacts with both hydrogen and oxygen in the formyl group. When the matrix containing a,a-2 was allowed to stand in the dark at 13 K, a gradual increase of the band due to anti-ketene 3 at the expense of 2 was observed. In contrast to its protio analogue, the deuterated carbene 2-d showed almost no changes either for the decay of 2-d or the growth of 3-d within the time range where the protio analogue appreciably decayed. These observations are interpreted in term of a quantum mechanical tunneling mechanism.

Journal Article↗
PubMedDate not supplied

Ankyrin binding to (Na+ + K+)ATPase and implications for the organization of membrane domains in polarized cells.

The interaction between membrane proteins and cytoplasmic structural proteins is thought to be one mechanism for maintaining the spatial order of proteins within functional domains on the plasma membrane. Such interactions have been characterized extensively in the human erythrocyte, where a dense, cytoplasmic matrix of proteins comprised mainly of spectrin and actin, is attached through a linker protein, ankyrin, to the anion transporter (Band 3). In several nonerythroid cell types, including neurons, exocrine cells and polarized epithelial cells homologues of ankyrin and spectrin (fodrin) are localized in specific membrane domains. Although these results suggest a functional linkage between ankyrin and fodrin and integral membrane proteins in the maintenance of membrane domains in nonerythroid cells, there has been little direct evidence of specific molecular interactions. Using a direct biological and chemical approach, we show here that ankyrin binds to the ubiquitous (Na+ + K+)ATPase, which has an asymmetrical distribution in polarized cells.

Animals↗
PubMed2005

A statherin and calcium enriched layer at the air interface of human parotid saliva.

Parotid saliva placed in 35-mm-diameter tissue culture dishes developed increasing surface viscoelasticity at the interface with air. A surface layer became visible with time, and was collected and analysed by protein electrophoresis which indicated that a single protein (pI 4.2; molecular mass approx. 6 kDa) predominated. Western blot analysis demonstrated that the major protein band reacted with an antiserum directed against the C-terminal of the calcium-binding salivary protein statherin. Matrix-assisted laser-desorption ionization-time-of-flight MS analysis gave a molecular mass of 5380 Da for the protein, corresponding to the molecular mass of statherin. Staining of film protein in electrophoresis gels was compared with statherin synthesized on a solid phase, and the mean statherin content of film formed from 1 ml of parotid saliva was measured as 7 nmol. The mean calcium content of the surface layer was 250 nmol. Surface rheology was greatly decreased in the presence of EDTA, whereas surface tension of saliva was unaffected by calcium chelation, suggesting that protein accumulated at the surface was unaffected. The results suggest that a layer rich in statherin forms at the interface of saliva and air, and that the surface rheology developed is dependent upon protein interactions mediated by calcium. The surface layer may enhance the function of saliva as a protective layer on the mucosal surfaces and teeth.

Air↗